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4 X 1.25 ml, for 500 reactions (20 μl)1. Aliquot reagents to avoid contamination and to avoid repeated freeze-thaw cycles.2. Primer concentration should not be high, a concentration of 100 nM to 300 nM of each primer usually gives the best results.3. A very effective way to get tight Ct among replicates is to reduce pipetting error, this can be achieved by: preparing amplicon specific pre-mix, using repeating pipet, and keeping pipetting volume in manufacture suggested range.4. For optimal results, it is recommended that the primers are 18-22 nucleotides in length with a Tm of 58°C-60°C and the size of target is about 100-250 bp.